Isolation of Inner Cell Mass for the Establishment of Human Embryonic Stem Cell (hESC) Lines

ABSTRACT

A method of establishing a cell line from the inner cell mass of a blastocyst, comprising: isolating a blastocyst having a zona pellucida, a trophectoderm, and an inner cell mass; creating an aperture in the blastocyst by laser ablation; isolating cells from the inner cell mass from the blastocyst through the aperture; and culturing the cells to establish a cell line, wherein the cells are cultured under feeder free conditions.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 10-226,711, filed Aug. 23, 2002, U.S. Pat. No. 7,294,508, whichclaims the benefit of priority of U.S. Provisional Application Ser. No.60/314,323 filed on Aug. 23, 2001, the disclosures of both of which areincorporated herein in their entirety by reference for all purposes.

FIELD OF THE INVENTION

The present invention relates to a method of isolation of inner cellmass (ICM) derived from blastocyst stage mammalian embryo forestablishing human embryonic stem cell (hESC) lines, using a non-contactdiode laser technique.

BACKGROUND OF THE INVENTION

The isolation of human stem cells offers the promise of a remarkablearray of novel therapeutics. Biologic therapies derived from such cellsthrough tissue regeneration and repairs as well as through targeteddelivery of genetic material are expected to be effective in thetreatment of a wide range of medical conditions. Efforts to analyze andassess the safety of using human stem cells in the clinical setting arevitally important to this endeavor.

Embryonic stem (ES) cells are the special kind of cells that can bothduplicate themselves (self renew) and produce differentiatedfunctionally specialized cell types. These stem cells are capable ofbecoming almost all of the specialized cells of the body and thus, mayhave the potential to generate replacement cells for a broad array oftissues and organs such as heart, pancreas, nervous tissue, muscle,cartilage and the like.

Stem cells have the capacity to divide and proliferate indefinitely inculture. Scientists use these two properties of stem cells to produceseemingly limitless supplies of most human cell types from stem cells,paving the way for the treatment of diseases by cell replacement. Infact, cell therapy has the potential to treat any disease that isassociated with cell dysfunction or damage including stroke, diabetes,heart attack, spinal cord injury, cancer and AIDS. The potential ofmanipulation of stem cells to repair or replace diseased or damagedtissue has generated a great deal of excitement in the scientific,medical and/biotechnology investment communities.

ES cells from various mammalian embryos have been successfully grown inthe laboratory. Evans and Kaufman (1981) and Martin (1981) showed thatit is possible to derive permanent lines of embryonic cells directlyfrom mouse blastocysts. Thomson et al. (1995 and 1996) successfullyderived permanent cell lines from rhesus and marmoset monkeys.Pluripotent cell lines have also been derived from pre-implantationembryos of several domestic and laboratory animal species such asbovines (Evans et al., 1990) Porcine (Evans et al., 1990, Notarianni etal., 1990), Sheep and goat (Meinecke-Tillmann and Meinecke, 1996,Notarianni et al., 1991), rabbit (Giles et al., 1993, Graves et al.,1993) Mink (Sukoyan et al., 1992) rat (Iannaccona et al., 1994) andHamster (Doetschman et al., 1988). Recently, Thomson et al (1998) andReubinoff et al (2000) have reported the derivation of human ES celllines. These human ES cells resemble the rhesus monkey ES cell lines.

ES cells are found in the ICM of the human blastocyst, an early stage ofthe developing embryo lasting from the 4th to 7th day afterfertilization. The blastocyst is the stage of embryonic developmentprior to implantation that contains two types of cells viz.

1. Trophectoderm: outer layer which gives extra embryonic membranes.

2. Inner cell mass (ICM): which forms the embryo proper.

In normal embryonic development, ES cells disappear after the 7th dayand begin to form the three embryonic tissue layers. ES cells extractedfrom the ICM during the blastocyst stage, however, can be cultured inthe laboratory and under the right conditions proliferate indefinitely.ES cells growing in this undifferentiated state retain the potential todifferentiate into cells of all three enthryonic tissue layers.Ultimately, the cells of the inner cell mass give rise to all theembryonic tissues. It is at this stage of embryogenesis, near the end offirst week of development, that ES cells can be derived from the ICM ofthe blastocyst.

The ability to isolate ES cells from blastocysts and grow them inculture seems to depend in large part on the integrity and condition ofthe blastocyst from which the cells are derived. In short, theblastocyst that is large and has distinct inner cell mass tends to yieldES cells most efficiently. Several methods have been used for isolationof inner cell mass (ICM) for the establishment of embryonic stem celllines. The most common methods are as follows:

1. Natural Hatching of the Blastocyst:

In this procedure blastocyst is allowed to hatch naturally after platingon the feeder layer. The hatching of the blastocyst usually takes placeon day 6. The inner cell mass (ICM) of the hatched blastocyst developsan outgrowth. This outgrowth is removed mechanically and is subsequentlygrown for establishing embryonic stem cell lines. However, thisprocedure has few disadvantages. First, trophectoderm cells proliferatevery fast in the given culture conditions and many times, suppress theoutgrowth of inner cell mass. Second, while removing the outgrowth ofthe inner cell mass mechanically, there is a chance of isolatingtrophectoderm cells. Third, the percentage of blastocysts hatchingspontaneously in humans is very low.

2. Microsurgery:

Another method of isolation of inner cell mass is mechanical aspirationcalled microsurgery. In this process, the blastocyst is held by theholding pipette using micromanipulator system and positioned in such away that the inner cells mass (ICM) is at 9 o'clock position. The innercell mass (ICM) is aspirated using a bevel-shaped biopsy needle and isinserted into the blastocoel cavity. This procedure too isdisadvantageous as the possibility to isolate the complete inner cellmass is low and many times cells get disintegrated. It is a very tediousprocedure and may cause severe damage to the embryo. The operation atthe cellular level requires tools with micrometer precision, therebyminimizing damage and contamination.

3. Immunosurgery:

Immunosurgery a commonly used procedure to isolate inner cell mass(ICM). The inner cell mass (ICM) is isolated by complement mediatedlysis. In this procedure, the blastocyst is exposed either to acidtyrode solution or pronase enzyme solution in order to remove the zonapellucida (shell) of blastocyst. The zona free embryo is then exposed tohuman surface antibody for about 30 min to one hour. This is followed byexposure of embryos to guinea pig complement in order to lyse thetrophectoderm. The complement mediated lysed trophectoderm cells areremoved from inner cell mass (ICM) by repeated mechanical pipetting witha finely drawn Pasteur pipette. All the embryonic stem cell linesreported currently in the literature have been derived by this method.However, this method has several disadvantages. First the embryo isexposed for a long time to acid tyrode or pronase causing deleteriouseffects on embryo, thereby reducing the viability of embryos. Second, itis a time consuming procedure as it takes about 1.5 to 2.0 hours.(Narula et al., 1996). Third, the yield of inner cell mass (ICM) perblastocyst is low. Fourth, critical storage conditions are required forantibody and complement used in the process. Last, it involves the riskof transmission of virus and bacteria of animal origin to humans, asanimal derived antibodies and complement are used in the process. Inthis process, two animal sera are used. One is rabbit antihumanantiserum and the other is guinea pig complement sera.

The human cell lines studied to date are mainly derived by using amethod of immunosurgery, where animal based antisera and complement wasused.

Other possible disadvantages of the existing cell lines are as follows:

1. Use of feeder cells for culturing the human embryonic stem cell(hESC) lines produces mixed cell population that require the Embryonicstem cells (ESC) to be separated from feeder cell components and thisimpairs scale up.

2. Embryonic stem cells (ESC) get contaminated by transcripts fromfeeder cells and cannot be used on a commercial scale. It can be usedonly for research purposes.

Geron established a procedure where human Embryonic Stem Cell (hESC)line was cultured in the absence of feeder cells (XU et.al 2001). ThehESC were cultured on an extracellular matrix in a conditioned mediumand expanded in this growth environment in an undifferentiated state.The hESC contained no xenogenic components of cancerous origin fromother cells in the culture. Also, the production of hESC cells and theirderivatives were more suited for commercial production. In this process,there was no need to produce feeder cells on an ongoing basis to supportthe culture, and the passaging of cells could be done mechanically.However, the main disadvantage of this procedure is that the inner cellmass (ICM) is isolated by immunosurgery method, wherein the initialderivation of Embryonic Stem Cells is carried out using feeder layercontaining xenogenic components. This raises the issue of possiblecontamination with animal origin viruses and bacteria.

In order to simplify the procedure of inner cell mass isolation and tomake it safe, the scientists of the present invention have come out witha novel method of isolation of the inner cell mass using a non-contactlaser, wherein, the use of animal based antisera and complement havebeen eliminated.

Use of Laser Technique in Assisted Reproduction:

With the advent of assisted reproductive technologies (ART), severalmethods have been used for improving fertilization, facilitatingblastocyst hatching (Cohen et al, 1990) and performing blastomere biopsy(Tarin and Handyside, 1993). Commonly used methods are chemical (Gordonand Talansky 1986), mechanical (Depypere et al., 1988) and laser(Feichtinger et al., 1992) so as to produce holes in the zona pellucida(Gordon, 1988). Recently, an infrared 1.48 μm diode laser beam focusedthrough a microscope objective was shown to allow rapid, easy andnon-touch microdrilling of mouse and human zona pellucida and highdegree of accuracy was maintained under conventional culture conditions(Rink et al., 1994). The drilling effect was shown due to a highlylocalized heat-dependent disruption of the zona pellucida glycoproteinmatrix (Rink et al., 1996). Contrary to the detrimental effect oncompacted mouse embryos induced by the 308 nm xenon-chlorine excimerlaser (Neev et al., 1993), the drilling process in the infrared regiondid not affect embryo survival in mice (Germond et al., 1995) or inhumans (Antinori et al., 1994).

Currently, lasers are being investigated as a tool to aid fertilizationand in assisted hatching. Recent reports show that use of 1.48 μm diodelaser for microdrilling mouse zona pellucida is highly safe and does notaffect neuro-anatomical and neurochemical properties in mice and alsoimproves fertilization (Germond et al., 1996). Obruca and colleaguesfirst reported the success of laser-assisted hatching in human IVF in1994. In this study, a 20- to 30-micron hole was made in the zonapellucida (ZP) when the embryos were at the two- to four-cell stage, andembryos were transferred immediately. Patients with previous IVFfailures from two separate centers were included in this study. Therewas a higher implantation rate per embryo in the laser-assisted hatchinggroup (14.4%) versus the control group (6%). Pregnancy rates pertransfer were also improved (40% versus 16.2%).

In a separate study, Er:YAG laser was used to thin the ZP of embryosderived from patients undergoing repeated IVF. Using a laser forthinning the ZP, embryologists are able to achieve accurate reduction ofthe ZP by 50%, which is very difficult with acidic Tyrode's solution.Presence of Acid Tyrode's solution near the embryo may also bedetrimental. The rate of clinical pregnancies in the laser-hatched groupwas 42.7%, as compared to 23.1% in the control unhatched group. Sincethis data looked promising, the indication of laser-assisted hatchingwas extended. Women undergoing IVF for the first time yielded 39.6%clinical pregnancy rate in the laser-treated group versus a 19% rate inthe control unhatched group (Parikh et al 1996).

During the last decade there has been ongoing research on the isolationof inner cell mass (ICM), as it is useful in establishing embryonic stemcell lines which in turn have the ability to develop into most of thespecialized cells in the human body including blood, skin, muscle andnerve cells. They also have the capacity to divide and proliferateindefinitely in culture.

The present invention involves the isolation of inner cell mass (ICM),using laser ablation technique without undergoing the cumbersomeprocedure of immunosurgery. Hence, in the present invention, the use ofanimal derived antibodies or sera are eliminated and the procedure issafe, simple, rapid, and commercially viable.

The present invention, obviates the shortcomings associated with theconventional methods of isolation of inner cell mass (ICM). The innercell mass (ICM) isolated by the present invention is found to be intactwithout causing any destruction or damage to the cells. The presentinvention thus provides a quick reliable and non-invasive method forisolation of inner cell mass (ICM). It also completely ruptures thetrophectoderm thereby minimizing the contamination of inner cell mass(ICM), thus ensuring the purity of inner cell mass (ICM).

REFERENCES

1. Antinori S, Versaci C, Fuhrberg P et al (1994). Seventeen live birthafter the use of erbium-yytrium aluminum garnet laser in the treatmentof male factor infertility. Hum Reprod. 9: 1891-1896.

2. Cohen J, Elsner C, Kort H et al (1990). Impairment of the hatchingprocess following IVF in the human and improvement of implantation byassisting hatching using micromanipulation. Hum Reprod. 5: 7-13

3. Depypere H T, McLaughlin K J, Seamark R F et al (1988). Comparison ofzona cutting and zona drilling as techniques for assisted fertilizationin the mouse. J. Reprod Fertil. 84: 205-211.

4. Doetschman T, Williams P and Maeda N (1988) Establishment of hamsterblastocyst derived embryonic stem (ES) cell. Developmental Biology 127:224-227.

5. Evans M J and Kaufman M H (1981). Establishment in culture ofpluripotential cells from mouse embryo. Nature 292: 154-156.

6. Evans M J, Notarianni E, Laurie S and Moor R M (1990) Derivation andpreliminary characterization of pluripotent cell lines from porcine andbovine blastocyst. Theriogenology 33: 125-128.

7. Feichtinger W, Strohmer H, Fuhrberg P et al (1992). Photoablation ofoocyte zona pellucida by erbium-yag laser for in-vitro fertilization insevere male infertility. Lancet. 339, 811.

8. Gordon J W (1988). Use of micromanipulation for increasing theefficiency of mammalian fertilization in vitro. Ann. N.Y. Acad. Sci.541: 601-613.

9. Gordon J W and Talansky B E (1986). Assisted fertilization by zonadrilling: a mouse model for correction of oligospermia. J. Exp Zool.239: 347-354.

10. Germond M, Nocera D, Senn A. Rink K. et al (1995). Microdissectionof mouse and human zona pellucida using 1.48 microns diode laser beam:efficacy and safety of the procedure. Fertil Steril. 64: 604-611.

11. Germond M, Nocera D, Senn A, Rink A et al (1996). Improvedfertilization and implantation rates after non-touch zona pellucidamicrodrilling of mouse oocytes with a 1.48 micron diode laser beam. HumReprod. 11: 1043-1048

12. Giles J R, Yang X, Mark X and Foot R H (1993). Pluripotency ofcultured rabbit inner cell mass cells detected by isozyme analysis andeye pigmentation of fetus following injection into blastocysts ormorula. Molecular Reproduction and Development 36: 130-138.

13. Graves K H and Moreadith R W (1993). Derivation and characterizationof putative pluripotential embryonic stem cells from pre-implantationrabbit embryo. Molecular reproduction and Development 36: 424-433.

14. Iannaccone P M, Taborn G U, Garton R L et al (1994). Pluripotentembryonic stem cells from the rat are capable of producing chimeras.Developmental Biology 163:288-292.

15. Martin G R (1981) Isolation of pluripotent cell lines from earlymouse embryos cultured in medium conditioned with teratocarcinoma stemcells. Proceeding of National Academy of Sciences USA 72: 1441-1445.

16. Meinecke-Tillmann S and Meinecke B (1996). Isolation of ES like celllines from ovine and caprine pre-implantation embryo. J Animal Breedingand Genetics 113: 413-426.

17. Narula A, Taneja, Totey S M (1996) Morphological cells totrophectoderm inner cell mass of in vitro fertilized andparthenogenetically developed buffalo embryo: the effect of IGF-1. Mol.Reprod. Dev. 44(3):343-51.

18. Neev J, Gonzales A, Lucciardi F et al (1993). Opening of the mousezona pellucida by laser without a micromanipulator. Hum Reprod. 8:939-944.

19. Obruca A, Strohmer H, Sakkas D (1994). Use of laser in assistedfertilization and hatching. Hum Reprod. 9:1723-1726.

20.Parikh F R, Kamat S A, Nadkarni S et al (1996). Assisted hatching inan in vitro fertilization program. J Reprod Fertil Suppl 50: 121-125

21. Reubinoff B E, Per M F, Fong C Y, Trounson A and Bongso A (2000)Embryonic stem cell lines from human blastocysts: Somaticdifferentiation in vivo. Nat Biotechnol. 18:299-304.

22. Rink K, Delacretaz G, Salathe R P et al (1994). Proceedings SPIE.2134A, 412-422.

23. Rink K, Delacretaz G, Salathe R P et al (1996). Non-contactmicrodrilling of mouse zona pellucida with an objective delivered 1.48microns diode laser. Lasers Surg Med. 18:52-62.

24. Sukoyan M A, Golublitsa A N, Zhelezova A I et al (1992) Isolationand cultivation of blastocyst derived stem cell lines from AmericanMink. Molecular Reproduction and Development 33: 418-431.

25. Tarin J J and Handyside A H (1993). Embryo biopsy strategies forpreimplantation diagnosis. Fertil Steril 59:943-952.

26. Thomson J A, Itskovitz-Eldor J, Shapiro S S. et al. (1998).Embryonic stem cell lines derived from human blastocyst. Science 282:1145-1147.

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OBJECTS OF THE INVENTION

1. It is an object of the present invention, to develop a process ofisolation of inner cell mass, using laser ablation technique, withoutundergoing the cumbersome procedure of immunosurgery.

2. It is another object of the present invention to isolate ICM usinglaser ablation technique without using any animal generated antibodiesand sera, thereby preventing the possibility of transmission of animalorganism to human and thus can be used on commercial scale.

3. It is another object of the present invention to isolate inner cellmass (ICM) from blastocyst stage of a mammalian embryo using anon-contact diode laser.

4. It is another object of the present invention to isolate inner cellmass (ICM) by simple, shorter and easily feasible way withoutaffecting/destroying the inner cell mass (ICM).

5. It is still another object of the present invention to ensure thepurity of inner cell mass (ICM) by rupturing completely trophectodermthereby minimising the contamination of inner cell mass (ICM).

6. It is still another object of the present invention to isolate innercell mass (ICM) of high yield and purity as compared to the inner cellmass (ICM) isolated by the conventional methods.

These and other objects of the invention will become more readilyapparent from the ensuing description.

DETAILS OF INVENTION

The present invention relates to isolation of inner cell mass, usinglaser ablation of zona pellucida (ZP) and trophectoderm (TE) andaspiration of inner cell mass for establishing embryonic stem celllines. In the present invention, the non contact diode laser used ishighly accurate and reliable tool for cellular microsurgery. The systemincorporates the latest in fiber optic technology to provide the mostcompact laser system currently available. The 1.48 .mu.m diodenon-contact Saturn Laser System is mounted/implanted via theepifluorescence port to inverted microscope fitted withmicromanipulators. A pilot laser is used to target the main ablationlaser and a series of LEDs inform the user when the laser is primed andis ready to fire. A two-second-operation window is used to reduce thepossibility of accidentally firing the laser. The spot diameter of thelaser can be varied according to the hole size required.

Couples undergoing in vitro fertilization (IVF) treatment voluntarilydonate surplus human embryos. These embryos are used for researchpurposes after taking the written, voluntary consent from these couples.In the present invention, blastocyst stage embryos are taken for theisolation of inner cell mass. The blastocyst is placed in a 35 mmpetridish in a 50 micro litre droplets of Ca++/Mg++ free embryo biopsymedium and is covered with mineral oil. The micromanipulator is set upto perform the embryo biopsy procedure. The blastocyst is placed inembryo biopsy medium and the petridish containing the blastocyst isplaced on the heating stage of the microscope. The blastocyst ispositioned at the center of the field. The blastocyst is immobilized onto the holding pipette in such a way that the inner cell mass is at 3o'clock position. The zona pellucida and trophectoderm close to innercell mass is positioned on the aiming spot of the laser beam. A smallportion of zona pellucida and trophectoderm is laser ablated. Biopsypipette is then gently inserted through the hole in the zona pellucidaand trophectoderm and the inner cell mass is gently aspirated. Afterisolation of the complete inner cell mass, the cells are given severalwashes with embryonic stem cell (ESC) medium. The cells are then platedon to feeder layer with embryonic stem cell medium for establishingembryonic stem cell lines. The embryonic stem cells were thencharacterized for cell surface markers such as SSEA-1, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, OCT-4 and alkaline phosphatase. The embryonic stemcell lines are also karyotyped.

a) Development of Blastocyst in Vitro:

Institutional Ethics Committee approval has been obtained beforeinitiation of this study. Prior written consent was taken fromindividual donor for the donation of surplus embryos for this studyafter completion of infertility treatment.

Protocol generally used for infertility patients for obtaining viableembryo is as follows:

The ovarian superovulation began with gnRH agonist analog suppressiondaily starting in the mid-luteal phase and administered in doses of500-900 mgs for about 9-12 days. Ovarian stimulation was started afteradequate ovarian suppression with human menopausal gonadotropins (hMG)or recombinant follicle stimulating hormone (FSH) (Gonal-F, Recagon) inappropriate doses depending on the age and ovarian volume. The dose wasalso adjusted as necessary to produce controlled ovarian stimulation.Serum beta-estradiol (E2) measurements were carried out as required.Vaginal ultrasound was performed daily from cycle day 7 onward. HumanChorionic gonadotropin 5000-10000 I.U. was administered when three ormore follicles were at least 17 mm in largest diameter. Transvaginalaspiration was performed 34-36 h later. Oocytes were then subjected tointracytoplasmic sperm injection.

A glass holding pipette 40-60 .mu.m in diameter was used to secure theegg. Motile sperm were placed in a drop of polyvinyl pyrolidone (PVP)solution and overlaid with mineral oil. An injection needle with anouter diameter of roughly 5-6 μm and inner diameter 3-4 μm was used topierce the zona pellucida at about 3 o'clock position. The selectedspermatozoon was immobilized by cutting the tail with the injectionmicropipette. The holding pipette secured the oocyte and spermatozoonwas injected directly into the center of the oocyte.

Oocytes were checked after 16-18 hours of culture for fertilization. Atthis point the fertilized oocyte had pro-nuclei (also called one cellembryo). One-cell embryos were then transferred into pre-equilibratedfresh ISM-1 medium and incubated at 37° C. in a 5% CO₂ in air. The nextday embryos were transferred into ISM-2 medium. Every alternate dayembryos were transferred into fresh ISM-2 medium. From day 5 onwardembryos were checked for the blastocyst development. After the treatmentis over, the surplus blastocysts were donated by the couples for thisresearch work.

b) Setting up of the Laser:

The present invention relates to describing a unique method for innercell mass isolation for establishment of embryonic stem cells using thenon-contact diode laser. The laser is highly accurate and reliable toolfor cellular microsurgery. The system incorporates the latest in fiberoptic technology to provide the most compact laser system currentlyavailable. The 1.48 μm diode non-contact Saturn Laser System was mountedvia the epifluorescence port to Zeiss inverted microscope fitted withmicromanipulators.

A pilot laser was used to target the main ablation laser and a series ofLED's, inform the user when the laser is primed and ready to dischargethe laser beam. A two-second-operation window was used to reduce thepossibility of accidentally firing the laser. The spot diameter of thelaser can be varied according, to the ablation size required.

c) Laser Ablation and Isolation of inner Cell Mass.

The blastocyst stage embryo was individually placed in a 50 μl drop ofbiopsy medium (Ca⁺⁺/Mg⁺⁺ free) in a 35-mm petri dish. The embryo wasimmobilized on to the holding pipette in such a way that the inner cellmass remained at 3 o'clock position and the zona pellucida andtrophectoderm close to inner cell mass positioned on the aiming spot. Acontinuous 1.48 μm diode laser was used to aperture the Zona Pellucida(ZP), which is a glycoprotein layer protecting the oocyte. At thiswavelength, the hole was induced by a local thermo-dissolution of theglycoprotein matrix. Once the zona pellucida was dissolved,trophectoderm cells were ablated by giving 3 pulses to cause photolysis.After ablation of both zona pellucida and trophectoderm, the aspirationpipette was introduced through laser-ablated hole and ICM was removed bygentle aspiration, having an internal diameter of 30-35 microns.

d) Culturing of Human Embryonic Stem Cells (hESC)

Prior to culturing, the aspirated ICM was washed thoroughly in ESmedium, which medium was found to be preferred for isolation ofembryonic stem cell lines. Given below is the procedure when theinvention was carried out using feeder layer. In this process, the innercell mass was cultured in 96 well plate in the presence of mouseinactivated embryonic fibroblast feeder layer. Embryonic fibroblastfeeder layer was preferably obtained from 12.5 to 13.5 day old C57BL/6mice or C57L/6XSJL F-1 mice or out bred CD1 mice or from human amnioticfluid-and used as a feeder layer. Embryonic fibroblast feeder layer wasinactivated by gamma irradiation (3500 rads). The mouse embryonicfibroblast feeder layer was cultured on 0.5% gelatin coated plate withES medium consisting of Dulbecco's modified Eagle's medium withoutSodium pyruvate with high glucose contain (70-90%), Fetal bovine serum(10-30%), beta-mercaptoethanol (0.1 mM), non-essential amino acids (1%),L-Glutamine 2 mM, basic fibroblast growth factor (4 ng/ml). Inner cellmass was then plated on mouse inactivated embryonic fibroblast. After4-7 days, ICM derived masses were removed from outgrowth with sterilefire polished pipette and were dissociated mechanically and plated onfresh feeder cells. Further dissociation was carried out with 0.5%trypsin-EDTA supplemented with 1% chicken serum.

Established cell lines were karyotyped and characterized for severalsurface markers such as SSEA-1, SSEA-3, SSEA-4, OCT-4, Alkalinephosphatase, TRA-1-81, TRA-1-60 as described by Thomson et al., (1998),Reubinoff et al., (2000).

EXAMPLES

The following examples are intended to illustrate the invention but donot limit the scope thereof.

Example 1

Total of 24 blastocyst stage human embryos were used for the isolationof inner cell mass. Embryos were washed several times in blastocystculture medium (ISM-2 medium, Medicult, Denmark). Individual blastocystwas then placed in the 50 μl drop of Ca++/Mg++ free embryo biopsy medium(EB 10 medium, Scandinavian). Micro drops were covered with mineral oil.Micromanipulator was set up. A glass holding pipette with outer diameter75 μm and inner diameter 15 μm was used to secure the embryo. Biopsypipette with an outer diameter of roughly 49 μm and inner diameter 35 μmwas used for aspiration of inner cell mass. A pilot laser was used totarget the main ablation laser. Embryo was immobilized on to the holdingpipette in such a way that inner cell mass remained at 3 o'clockposition and the zona pellucida and trophectoderm close to inner cellmass positioned to the aiming spot. The hole was induced by a localthermo-dissolution of the zona. Trophectoderm cells were ablated bygiving 3 pulses to cause photolysis. After ablation of both the zonapellucida and trophectoderm, the biopsy pipette was introduced throughlaser ablated hole and inner cell mass was removed. Inner cell mass wasthen washed several times in ES medium and placed in 96 well dish in thepresence or absence of feeder cells. The following data is presented inthe tabular form. TABLE 1 Summary of hESC lines developed using Laserablation Technique of the present invention with the use of mouse feedercells. With mouse feeder cells No. of blastocysts used Total inner cellNo. of No. of ES cell lines for laser ablation mass removed ICM usedestablished 24 18 14 4

Similarly, an experiment was conducted with conventional method ofisolation of inner cell mass i.e. using immunosurgery and may bereported as follows:

Example 2

The objective was to determine efficiency of isolation of inner cellmass with conventional method, i.e. immunosurgery, and compared withnewly invented laser ablated method.

Twenty-one blastocyst stage human embryos were used for isolation ofinner cell mass. Embryos were washed several times with blastocystculture medium (ISM-2 medium) and followed by ES medium. Individualblastocyst stage embryo was then placed in 50 μl microdrops of 1:50anti-human antibody (Sigma) for 30 minutes at 37° C. and 5% CO₂ in air.Blastocyst stage embryos were then washed four times after incubationwith ES medium. Blastocysts were then again placed in 50 μl ofmicrodrops of guinea pig complement at the concentration of 1:10 for 10minutes at 37° C. and 5% CO₂ in air. After incubation blastocyst stageembryos were washed several times in ES medium using fine bore glasspipette in order to remove trophectoderm. Isolated inner cell mass wasthen washed with ES medium and cultured in 96 well plate in the presenceor absence of feeder cells. Data are presented in the table: TABLE 2Summary of hESC lines developed using immunosurgery with/without the useof mouse feeder cells. With mouse feeder Without mouse No. of cellsfeeder cells blastocyst Total No. No. No. used inner of of ES of No. ofES for laser cell ICM cell lines ICM cell lines ablation mass removedused established used established 21 14 12 3 2 0

Although the isolation of inner cell mass using both the methods did notshow any significant difference, however, of isolation of inner cellmass by laser ablation has distinct advantage. This method willeliminate the use of antibodies and sera of animal origin. Isolation ofinner cell mass by laser ablation method can be further cultured in thepresence or absence of feeder layer. However, culturing of inner cellmass in a feeder free condition will further eliminate the possibilitiesof contamination of ES cell lines with animal viruses or bacteria andcan be commercially utilized for human transplantation studies. In thecurrent experiments, efforts were made to establish ES cell line in theabsence of feeder cells.

A preferred embodiment of the invention is illustrated in theaccompanying drawings.

FIG. 1(a) to 1(g) of the present invention, pertains to the isolation ofinner cell mass (ICM) from the blastocyst of one embryo and FIG. 2(a) to2(g) pertains to the isolation of inner cell mass (ICM) from theblastocyst of another embryo. FIGS. 3, 4, and 5 pertains to culturing ofICM on feeder cells at different stages.

FIG. 1 (a) is a scanned image of human blastocyst, secured with glassholding pipette such that the ICM is at 3 o'clock position.

FIG. 1 (b) is a scanned image wherein part of zona pellucida andtrophectoderm ablated with laser (arrow).

FIG. 1 (c) is a scanned image of aspiration pippette close to theblastocyst following zona and trophectoderm ablation.

FIG. 1 (d) is a scanned image of beginning of aspiration of ICM withaspiration pipette.

FIG. 1 (e) is a scanned image of large portion of ICM in the aspirationpipette during aspiration process.

FIG. 1 (f) is a scanned image of the ICM after removing from theblastocyst.

FIG. 1 (g) is a scanned image of the remaining trophectoderm and zonapellucida remaining after ICM isolation.

FIG. 2 (a) is a scanned image of another human blastocyst, secured withglass holding pipette such that the ICM is at 3 o'clock position.

FIG. 2 (b) is a scanned image of slight protrusion of inner cell massafter zona and trophectoderm is laser ablated.

FIG. 2 (c) is a scanned image of the aspiration pipette being positionclose to the ICM after ablating the zona and neighboring trophectodermcells with laser.

FIG. 2 (d) is a scanned image of ICM being aspirated with the aspirationpipette by gentle suction.

FIG. 2 (e) is a scanned image of large portion of ICM in the aspirationpipette.

FIG. 2 (f) is a scanned image of the ICM after removing from theblastocyst.

FIG. 2 (g) is a scanned image of the trophectoderm and zona pellucidaleft after the isolation of ICM from the blastocyst.

FIG. 3 (a) is a scanned image of isolated inner cell mass in cultureseeded on primary mouse embryonic fibroblast feeder cells (day 3).

FIG. 3 (b) is a scanned image of isolated inner cell mass in culture onprimary mouse embryonic fibroblast feeder cells (day 7).

FIG. 4 is a scanned image of isolated ICM in culture on the primarymouse embryonic fibroblast feeder cells (day 5) another embryo.

FIG. 5 is a scanned image of embryonic stem cell line derived from innercell mass isolated by laser ablation method.

One skilled in the art will appreciate that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned therein above. The instant invention has been shown anddescribed herein in what is considered to be the most practical andpreferred embodiment. It is recognized that departures may be made fromwithin the scope of the invention. It is to be understood that theinvention is not limited to the particulars disclosed and extends to allequivalents within the scope of the scope of the claims.

1-20. (canceled)
 21. A method of establishing a cell line from the innercell mass of a human blastocyst, comprising: (a) isolating a blastocysthaving a zona pellucida, a trophectoderm, and an inner cell mass; (b)creating an aperture in the blastocyst by laser ablation; (c) isolatingcells from the inner cell mass from the blastocyst through the aperture;and (d) culturing the cells to establish a cell line, wherein the cellsare cultured under feeder free conditions.
 22. The method of claim 21,wherein the aperture is through the zona pellucida.
 23. The method ofclaim 21, wherein the aperture is through the zona pellucida and thetrophectoderm.
 24. The method of claim 21, wherein the laser ablation isachieved using a non contact diode laser.
 25. The method of claim 24,wherein the non contact diode laser is a continuous 1.48 μm diode laser.26. The method of claim 21, wherein cells from the inner cell mass areisolated by aspiration.
 27. The method of claim 21, wherein isolatingcells from the inner cell mass is carried out in the absence of animalgenerated antibodies and sera.
 28. The method of claim 21, furthercomprising a micromanipulator system comprising a microscope with aheating stage, a holding pipette, an aspiration pipette, and an airsyringe, wherein the isolated blastocyst of step (a) is placed on theheating stage, the micromanipulator system is adjusted so that theblastocyst is at the center of the microscope field, and the blastocystis secured with the holding pipette by suction through the air syringe,so that the inner cell mass is opposite the holding pipette.
 29. Themethod of claim 21, wherein the feeder free conditions comprise anextracellular matrix.
 30. The method of claim 29, wherein the feederfree conditions further comprise conditioned medium.
 31. The method ofclaim 21, wherein the isolated blastocyst is the product of in vitrofertilization.
 32. A method of establishing a human embryonic stem cellline, comprising: (a) isolating a human blastocyst comprising an innercell mass; (b) creating an aperture in the blastocyst by laser ablation;(c) isolating cells from the inner cell mass of the blastocyst throughthe aperture; and (d) culturing the cells under feeder free conditionsto obtain an isolated human embryonic stem cell line.
 33. The method ofclaim 32, wherein the aperture is through the zona pellucida and thetrophectoderm.
 34. The method of claim 32, wherein the laser ablation isachieved using a non contact diode laser.
 35. The method of claim 32,wherein the cells of the inner cell mass are cultured to produce innercell mass-derived cell masses.
 36. The method of claim 35, wherein theinner cell mass-derived cell masses are dissociated and re-plated on anextracellular matrix.
 37. The method of claim 32, wherein cells of theinner cell mass are isolated by aspiration.
 38. The method of claim 32,wherein the human embryonic stem cell line is established in the absenceof animal generated antibodies and sera.
 39. The method of claim 32,wherein the feeder free condition comprises plating the cells of theinner cell mass on an extracellular matrix.
 40. A method of establishinga human embryonic stem cell line, comprising: (a) isolating cells of aninner cell mass from a blastocyst by creating an aperture in theblastocyst by laser ablation and removing cells of the inner cell massfrom the blastocyst through the aperture; (b) culturing the cells of theinner cell mass to produce inner cell mass derived masses; and (c)culturing the inner cell mass derived masses under feeder freeconditions to produce an isolated human embryonic stem cell line. 41.The method of claim 40, wherein the feeder free conditions comprise anextracellular matrix.
 42. The method of claim 40, further comprisingmechanically dissociating the inner cell mass derived masses of step (b)and re-plating the mechanically dissociated cells of the inner cell massderived masses under feeder free conditions.